With development of genetic recombination techniques, various protein formulations have become available in stable supply. Specifically, in recent years, various therapeutic antibodies with higher selectivity than that of conventional medicinal products have been developed by genetic recombination techniques and have entered clinical trials.
For drug products containing a biologically active protein produced by such genetic recombination techniques, it is necessary to remove host cell-derived proteins (host cell proteins) and DNA, resin ligand fragments which are one of the raw materials in purification, and aggregates or fragments derived from the protein of interest. Furthermore, to ensure safety of the drug products against viruses, the purification step must be shown to have a sufficient ability to remove or inactivate viruses. Currently, the World Health Organization (WHO) indicates that the acceptable amount of DNA in a biological medicinal product is 100 pg DNA/dose or less. Furthermore, the World Health Organization (WHO) indicates that regarding viruses, if the presence of retrovirus-like particles is observed in the cultured solution, the drug product may contain no more than one virus particle per 106 doses after taking into account the ability to remove or inactivate retroviruses in the purification step. Generally, to meet this criterion, impurities are removed by treating the aqueous cultured medium containing the bioactive protein obtained from the host cells with affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydroxyapatite chromatography, or hydrophobic interaction chromatography, or a combination thereof. Furthermore, development of new purification ligands has advanced in recent years, and multimodal chromatography that has two functions of both the ion-exchanging action and hydrophobic interaction is also used for purification.
In particular, when the bioactive protein is an antibody that can be obtained by using mammalian cells as the host, it is purified by treatment with the Protein A or Protein G affinity column chromatography by utilizing the property of Protein A or Protein G to bind to the Fc region of IgG, followed by various chromatography methods.
For example, in Japanese Patent Application Kohyo Publication No. (JP-A) H05-504579 (unexamined Japanese national phase publication corresponding to a non-Japanese international publication) (Patent Document 1), an antibody-containing aqueous cultured medium obtained from a mammalian cell culture was subjected to a protein A or protein G column chromatography to absorb the antibody to the column, the antibody was then eluted using an acidic solution (citric acid at a concentration of approximately 0.1 M at pH 3.0-3.5), and the resulting acidic eluate was purified by subsequent ion-exchange column chromatography and size exclusion column chromatography.
In order to increase the blood retention or in vivo kinetics, amino acid substitution techniques for controlling the isoelectric point (pI) of an antibody, specifically the technique of altering amino acid residue(s) exposed on the surface of an antibody to control the pI of the antibody (WO 07/114319 (Patent Document 2)) are known. The isoelectric point (pI) of a native antibody is in the range of approximately 7.5-9.5 and is a relatively high pI. Lowering the pI by modifying the amino acid residues of such an antibody is expected to prolong the plasma retention and half-life of the antibody, and this will lead to reduction in the amount of antibody administered as a drug and extension of the administration intervals.
However, until now, there has been no investigation on purification methods that are appropriate for low-pI antibodies which do not exist in nature, or examination of issues specific to low-pI antibodies in the purification process. Therefore, nothing is known with respect to purification methods that are suitable for such low-pI antibodies.